Peptide Libraries
Classically, the pharmaceutical industry has screened a wide variety of compounds derived from natural sources to yield potential drug candidates or lead compounds for the development of new drugs. These laborious screening efforts have relied on the random testing of a vast number of chemical entities. In recent years, various strategies have been adopted for the generation of libraries of compounds that are subsequently screened as a novel, rational approach to drug discovery and development.
It has become apparent that a variety of methodologies can be applied to the problem of generating a diverse group of candidate compounds, based on the known principles of peptide chemistry and/or molecular biology. Peptides are a convenient class of molecules for the generation of combinatorial libraries, since they are composed of a finite set of amino acid building units, which can be efficiently assembled either by chemical synthesis or transcription/translation of DNA. Combinatorial libraries are discussed by Gallop et al., J. Med. Chem., 37, 1233-1251 (1994); Gordon et al., J. Med. Chem., 37, 1385-1401 (1994); Pinilla et al., Biopolymers (Peptide Science), 37, 221-240, (1995); and Lebl et al., Biopolymers (Peptide Science), 37, 177-198 (1995). The set of amino acid building units can include only the naturally encoded amino acids, when the libraries are encoded by oligonucleotides on a plasmid, phage, or any other vector. This set can be expanded to include both D and L amino acids and/or non-natural amino acids in synthetic libraries.
Linear peptides suffer from several serious drawbacks as potential drugs, inasmuch as they are notoriously unstable in vivo, often lack high affinity of binding to their receptor, frequently lack selectivity to one kind of receptor, and generally have poor oral bioavailability. In efforts to overcome such problems, it is also possible to utilize the methodologies developed in connection with synthetic peptide libraries to generate collections of cyclic peptides, novel biopolymers and even novel branched oligomeric compounds, reviewed by Zuckermann, Current Opinion in Structural Biology, 3, 580-584 (1993).
One of the most significant synthetic technologies that facilitate the generation and screening of diverse chemical libraries is the resin-splitting method, which is a polymer supported multiple synthesis procedure that allows a high degree of control over the composition of a peptide mixture. Mixtures are generated by dividing a solid support into individual portions, and coupling a different amino acid to each portion, and then recombining the portions. These steps may be performed in an iterative fashion to provide the required degree of diversity.
Totally random libraries generated by these types of methods are disclosed in WO92/00091 and WO92/09300. Each individual bead will contain a unique peptide sequence, which can be probed for activity with a soluble receptor or antibody. Positive beads can be isolated and sequenced using Edman sequencing chemistry. WO92/00091 further discloses methods to provide selectively cleavable linkers between peptide and resin, such that part of the peptide can be liberated from the resin and assayed for activity in soluble form, while another part can be sequenced. In addition, it is also possible to generate random libraries in which each bead carries more than one peptide, by coupling of mixtures of amino acids to the beads, as disclosed by Hornik et al., Reactive Polymers, 22, 213-220 (1994).
Another methodology is disclosed by Geysen et al., J. Immunol. Meth., 102, 259-274 (1987), which involves the synthesis of peptides on derivatized polystyrene pins which are arranged in such a fashion that they correspond to the arrangement of wells in a 96-well microtiter plate. Individual chemical reactions can be performed in each well, thereby yielding individual peptides on each pin. The pins are typically probed using an enzyme linked immunoassay (ELISA) or radioimmunoassay (RIA), carried out in the microtiter wells, or the peptides may be released from the pins and tested in solution. The mimotope approach of Geysen et al. generates diverse peptides that are probed for activity in situ. The best dipeptide sequence is selected for elongation to diverse tripeptides, the best tripeptide is selected for elongation to a tetrapeptide and so on.
Ideally, chemistries that are amenable to combinatorial library synthesis would have the following characteristics: be polymer-supported to facilitate the resin splitting technique; be assembled in high yield with automatable chemistry; and allow the incorporation of a wide variety of chemical functionalities.
Cyclic Peptides
Cyclic peptides are generally recognized as possessing enhanced bioavailability due to increased metabolic stability, as well as a relatively constrained conformation when compared to the same sequence in a linear form. The enhanced metabolic stability should allow diminished doses at longer intervals. The restricted conformation should improve the drug selectivity, thereby potentially preventing side-effects. All of these properties are desirable in conjunction with the quest for new drug candidates.
The generation of libraries of cyclic peptides requires, in addition to any previously stated considerations, that the cyclization reaction be performed in a high yield and with a minimum of additional manipulations. Unfortunately, classical cyclization reactions are highly sequence dependent in terms of the expected yields, making the uniform cyclization of a peptide mixture unreliable.
Recent advances in the cyclization of peptides directly on the solid support have improved the synthetic procedure, and even allowed the automation of cyclization reactions based on known cyclization schemes. In the past, cyclizations were typically performed in solution under conditions of high dilution. Polymer-supported cyclizations can both avoid potential side reactions such as oligomerization and facilitate product purification. For example, on-resin cyclization methods have recently been used to prepare cyclopeptides with bridges formed of thioethers, disulfides, or lactams between two side chains, lactams between the amino terminus and a side chain, and lactams between the amino and carboxy termini (reviewed by Zuckermann, Current Opinion in Structural Biology, 3, 580-584 (1993).
The use of resin-bound cyclic peptides and free cyclic peptides in combinatorial libraries is disclosed in WO 92/00091. However, these cyclic peptides do not contain any conformationally constraining element, and in cases where cyclization is achieved, these peptides may still adopt a number of conformations and suffer many of the same shortcomings as linear peptides.
Cyclic semi-random peptide libraries, which are disclosed in WO 95/01800, are exclusively cyclic penta-peptide and hexa-peptide libraries containing one or more randomized amino acids and a conformationally constraining element in the form of an amino acid residue such as proline which fixes the beta turn angles of the adjacent amino acid residues. The advantages of such conformationally constraining elements is stressed by the inventors of this approach. However, inclusion of such elements via incorporation of a particular amino acid residue into the peptide sequence may have detrimental effects on those residues required for receptor recognition or other biological activity. Furthermore, in WO 95/01800, the cyclization reaction is merely another coupling reaction in which the terminal amino group of the linear peptide is coupled to the terminal carboxy group of the peptide.
Backbone Cyclized Peptides
Backbone cyclized peptides are generally known, as discussed, for instance, in Gilon et al., Biopolymers, 31, 745-750 (1991) and in EPO 564,739 A2 and EPO 564,739 A3. Such compounds have not been used for constructing libraries for screening purposes.
In addition, methods are known for combining amino acids and peptides. U.S. Pat. No. 5,010,175 describes another method of incorporating random amino acids into a peptide. According to that method, a mixture of amino acids is incorporated by coupling a mixture in which the individual amino acids are present in varying proportions depending upon their relative rates of reaction in the coupling, e.g., the amount of amino acid is inversely proportional to its rate of coupling.